This revised renewal proposal is designed to delineate novel gene regulatory mechanisms and downstream pathways involved in the control of muscle glucose utilization and exercise capacity relevant to the consequences of chronic caloric excess. Current dogma suggests that accumulation of skeletal myocyte lipid leads to insulin resistance caused by derangements within the insulin signaling pathway. We have shown during the current funding period that myocyte lipid accumulation, driven by chronic activation of the nuclear receptor peroxisome proliferator-activated receptor a (PPARa), is linked to reduced muscle glucose uptake and utilization, independent of derangements in the insulin signaling pathway. The metabolic phenotype of muscle-specific PPARa transgenic mice is similar to that of the obesity-related insulin resistant state including myocyte triacylglyceride (TG) accumulation, high fatty acid oxidation rates, glucose intolerance, and reduced exercise capacity. In striking contrast, transgenic lines for PPAR2, a muscle-expressed relative of PPARa, exhibit many of the metabolic benefits of exercise in the absence of training, including increased capacity for muscle glucose oxidation and an increase in slow-twitch muscle fibers. Transcriptional and metabolomic profiling results strongly suggest that whereas PPARa and PPAR2 regulate many overlapping gene targets involved in cellular fatty acid utilization, a subset is PPAR isotype-specific. For example, PPAR2 (but not PPARa) activates genes involved in muscle glucose uptake/oxidation and the slow-twitch muscle fiber program, the latter through a distinct microRNA network. We have also shown that the PPAR coactivator, PGC-1a, is necessary and sufficient for muscle mitochondrial function and biogenesis programs that are activated by exercise training. These results have led us to hypothesize that PPARa and PPAR2 control overlapping, as well as distinct, genes in skeletal muscle and that delineation of PPAR isotype-specific targets will unveil novel strategies to re-program skeletal muscle metabolism for the prevention and treatment of the consequences of obesity, such as insulin resistance. This hypothesis will be tested by: delineating the gene regulatory mechanisms involved in the re-programming of muscle glucose metabolism (Aim 1) and fiber type (Aim 2) by PPAR2 versus PPARa;conducting unbiased genomic and metabolomic interrogations to define PPAR2- and PPARa-specific target genes and metabolic pathways (Aim 3);and performing studies in vivo in mice to validate the actions and relevance of the candidate regulatory molecules and pathways downstream of PPAR2 in the control of muscle fuel metabolism, energetics, and exercise capacity under normal physiological conditions and in the context of diet-induced insulin resistance (Aim 4). In vivo validation of novel molecules, pathways, and metabolomic signatures downstream of PPAR2 will allow us to develop a prioritized list of new candidate therapeutic targets (and predictive biomarkers) for enhancing muscle fuel metabolism and function relevant to the ravages of caloric excess. PUBLIC HEALTH RELEVANCE: We are witnessing a pandemic in obesity-related diabetes. This project seeks to identify new molecules and metabolic pathways in muscle that could serve as novel therapeutic targets aimed at reducing the development of insulin resistance and progression to diabetes in the obese population. The proposed studies also show great promise for developing new therapeutic strategies to enhance skeletal muscle function in chronic debilitating disease states and with aging.